sign in sign up
<<
Home , Ketone bodies, Ketosis

ARTICLE IN PRESS

Prostaglandins, Leukotrienes and Essential Fatty Acids 70 (2004) 309–319

Review The therapeutic implications of bodies: the effects of in pathological conditions: , , redox states, resistance, and mitochondrial Richard L. Veech* Laboratory of Membrane Biochemistry and Biophysics, National Institutes of Alcoholism and Abuse, 12501 Washington Ave., Rockville, MD 20850, USA Received 10 August 2003; accepted 1 September 2003

Abstract

The effects of ketone body metabolism suggests that mild ketosis may offer therapeutic potential in a variety of different common and rare disease states. These inferences follow directly from the metabolic effects of ketosis and the higher inherent energy present in d-b-hydroxybutyrate relative to pyruvate, the normal mitochondrial fuel produced by leading to an increase in the DG0 of ATP hydrolysis. The large categories of disease for which may have therapeutic effects are: (1) diseases of substrate insufficiency or , (2) diseases resulting from free radical damage, (3) disease resulting from hypoxia. Current ketogenic diets are all characterized by elevations of free fatty acids, which may lead to metabolic inefficiency by activation of the PPARsystem and its associated uncoupling mitochondrial uncoupling . New diets comprised of ketone bodies themselves or their esters may obviate this present difficulty. Published by Elsevier Ltd.

1. Metabolic effects of ketone body metabolism that the effects of ketone metabolism in would mimic those in , which was not analyzed in this The therapeutic potentials of mild ketosis flow detailed manner for a number of technical reasons, most directly from a thorough understanding of their meta- prominently the inhomogeneous nature of the tissue and bolic effects, particularly upon mitochondrial redox its lack of quantifiable outputs. states and energetics and upon substrate availability. A detailed metabolic control strength analysis of The data on metabolic effects of ketone body metabo- glycolysis in heart under the four conditions led to lism presented here has been published previously [1,2]. several major conclusions [1]. Firstly, the control of flux It presents studies of the isolated working rat heart through the glycolytic pathway was context dependent perfused with 11 mM alone, glucose plus 1 mM and shifted from one enzymatic step to another acetoacetate and 4 mM d-b-hydroxybutyrate, gluco- depending upon the conditions. There was not one key se+100 nM insulin or the combination of glucose, ‘‘rate controlling’’ reaction, but rather control was ketone bodies and insulin. The isolated working distributed among a number of steps, including some perfused heart was studied because of the relative enzymes that were very close to equilibrium. The homogeneity of the tissue and the simplicity of its absence of a single dominant rate controlling step in a output, the number of parameters which could be pathway calls into question the assumptions on which accurately measured, particularly O2 consumption many pharmaceutical discovery programs have been relative to actual hydraulic work output of the heart. based [3]. Secondly, when perfused with glucose alone, In our analysis of disease states, it has been assumed there was consistent breakdown, whereas with addition of ketones, insulin or the combination, *Tel.: +1-301-443-4620; fax: 1-301-443-0930. glycogen synthesis occurred. Addition of either ketones E-mail address: [email protected] (R.L. Veech). or insulin, increased intracellular [glucose] and the

0952-3278/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.plefa.2003.09.007 ARTICLE IN PRESS 310 R.L. Veech / Prostaglandins, Leukotrienes and Essential Fatty Acids 70 (2004) 309–319 glycolytic intermediates in the first half of the glycolytic working perfused heart [2]. The proton gradient created pathway from 2 to 8 fold. Thirdly, addition of either by the redox energy of the respiratory chain then powers ketones or insulin leads to an increase in the measurable the transport of protons from cytosol back into hydraulic work of the heart, but a net decrease in the mitochondria through the F1 ATPase complex in an rate of glycolysis. Associated with the increase in efficient and reversible process [7,8]. For each electron hydraulic work and decrease in glycolytic rate addition pair transported up the respiratory chain, 3 ATPs are of ketones or insulin increased the free cytosolic [ATP]/ generated. Since the maximum energy available from the [ADP]  [Pi] ratio three to five fold and a similar change redox reactions of the chain is À178 kJ, the energy in the [phosphocreatine]/[creatine] ratio showing that available for the synthesis of each of the 3 ATPs can not both ketones or insulin increased the energy of the exceed À59.2 kJ/mol. The energy of the hydrolysis of 0 phosphorylation state of heart significantly compared to ATP, DGATP; in heart, and red cell under 6 perfusion with glucose alone. These data clearly show conditions ranged from À54 to À58 kJ/mol, implying that addition of either ketone bodies or insulin, that the overall process of electron transport and markedly improved the energy status of working oxidative phosphorylation is a remarkably efficient perfused heart. How ketone bodies could increase the process. hydraulic efficiency of heart by 28% could not be The electrons liberated from mitochondrial NADH explained by the changes in the glycolytic pathway by the NADH dehydrogenase complex or complex I, are alone, but rather by the changes that were induced carried within the mitochondria by co-enzyme Q, where in mitochondrial ATP production by ketone body they are transferred to cytochrome C, by CoQH2- metabolism. cytochrome C reductase, complex III. The difference The mitochondrial processes of ATP generation between the redox potential of the mitochondrial NAD derive their energy from the respiratory electron couple and the co-enzyme Q couple, DEQ=NADH; transport chain, where the electrons from substrates determines the energy of the proton gradient generated enter at different catalytic centers and travel up through by mitochondria. This in turn determines the energy of 0 various redox couples within the chain to ultimately hydrolysis of ATP, DGATP; which is generated by the + combine with H and O2 to form H2O. The respiratory mitochondrial F1 ATPase [2]. chain begins with the NADH multienzyme complex, The ketone bodies, acetoacetate and d-b-hydroxybu- whose substrate is the free mitochondrial NADH. The tyrate are in near-equilibrium with the free mitochon- redox potential of the free mitochondrial [NAD+]/ drial [NAD+]/[NADH] ratio in a reaction catalyzed by [NADH] couple is about À0.28 V [4] while that of the d-b-hydroxybutyrate dehydrogenase [9] It would also [O2]/[H2O] couple is +1.2 V [5]. The total energy appear that the [succinate]/[[fumarate] couple is in near available from the movement of electron up the equilibrium with the mitochondrial [Q]/[QH2] couple in respiratory chain is therefore determined by the a reaction catalyzed by succinate dehydrogenase [2]. difference between the variable redox potential of the When ketone bodies are metabolized in heart, the mitochondrial NAD couple and the O2 couple, which is mitochondrial NAD couple is reduced while the constant at all O2 concentrations and is given by mitochondrial Q couple is oxidized increasing the redox DG0 ¼ÀnFDE; span, DEQ=NADH; between site I and site III, making more energy available for the synthesis of ATP, and where n ¼ 2 electrons and F ¼ 96:485 kJ/mol/V. hence an increase in the DG0 of ATP hydrolysis. This in This means 2 electrons traveling up the electron turn is observable in the 28% increase in the hydraulic transport system in the redox reaction efficiency of the working perfused rat heart. þ 1 - þ The fundamental reason why the metabolism of NADHmito þ H þ 2O2 H2O þ NADmito ketone bodies produce an increase of 28% in the can yield À178 kJ/2 moles of electrons. hydraulic efficiency of heart compared with a heart The energy gained from mitochondrial electron metabolizing glucose alone is that there is an inherently transport is transferred to the pumping of protons from higher heat of combustion in d-b-hydroxybutyrate than the mitochondrial to cytosolic phase [6] at sites I, III and in pyruvate, the mitochondrial substrate which is the IV creating an electrochemical proton gradient between end product of glycolysis (Table 1). the two phases where the energy of the proton gradient If pyruvate were burned in a bomb calorimeter, it is would liberate 185.7 kcal/mole of C2 units, whereas the 0 þ þ d DG ½H cyto=½H mito combustion of -b-hydroxybutyrate would liberate 243.6, or 31% more calories per C unit than ¼ RT ln½Hþ =½Hþ þ FE ; 2 cyto mito mito=cyto pyruvate. Metabolizing d-b-hydroxybutyrate in per- where the major energy component is the electric fused working heart creates a 28% increase in the potential between the mitochondrial and cytosolic hydraulic efficiency of heart when compared to the phases, which ranges between À120 and À140 mV in metabolism of the end product of glycolysis, pyruvate. ARTICLE IN PRESS R.L. Veech / Prostaglandins, Leukotrienes and Essential Fatty Acids 70 (2004) 309–319 311

Table 1 In addition to their effects on mitochondrial ener- Heats of combustion of common non-nitrogenous energy substrates getics, the metabolism of ketone bodies has other effects o o Substrate DH kcal/mol DH kcal/mol C2 units with therapeutic implications. Of major significance is the ability of ketone bodies to increase the concentra- C H O Palmitate À2384.8 À298 18 32 2 tions of the metabolites of the first third of the C4H8O3 b HOButyrate À487.2 À243.6 C6H12O6 Glucose À669.9 À223.6 cycle. Next to causing the translocation of GLUT4 from C3H4O3 Pyruvate À278.5 À185.7 endoplasmic to plasma membrane, one of the most important acute metabolic effects of insulin, is the stimulation of the activity of the pyruvate dehydrogen- The mitochondrial processes of electron transport and ase multienzyme complex leading to an increased ATP synthesis appear to be capable of capturing this production of mitochondrial acetyl CoA, the essential inherent energy contained in the substrates being substrate for the . The metabolism of metabolized. ketone bodies or the addition of insulin to working The greater energy inherent in d-b-hydroxybutyrate perfused heart both results in increases of cardiac acetyl relative to pyruvate derives from the higher ratio of H–C CoA content 12–18 fold. This increase is associated with present in each molecule, 2 H per C in the case of d-b- increases of 2–8 fold in the cardiac content of citrate and hydroxybutyrate versus 1.3 H per C in the case of the other constituents of the citric acid cycle down to a- pyruvate. Put another way, the ketone body is more ketoglutarate, and including l-glutamate, an important reduced than pyruvate. One may then ask, why would redox partner of a-ketoglutarate and the mitochondrial there not be even more energy released if the heart were NAD couple. Citrate is an important precursor in the to metabolize the palmitate, which has even generation of cytosolic acetyl CoA for and acetyl more inherent energy available during combustion than choline biosynthesis, while l-glutamate is a necessary a ketone body. The reasons why the metabolism of fatty precursor for GABA synthesis in neural tissue. acids does not lead to even a greater increase in The metabolic effects of ketone bodies are of efficiency than does the metabolism of ketones are of particular relevance to brain metabolism, where Cahill two types: the architecture of the pathway of fatty acid and his colleagues have established that over 60% of the oxidation and the enzymatic changes induced by metabolic energy needs of brain can be supplied by elevation of free fatty acids. ketone bodies, rather than by glucose [11]. They have During b oxidation, only half of the reducing further established that mild ketosis with blood levels of equivalents released enter the respiratory chain at 5–7 mM is the normal physiological response to NADH dehydrogenase while the other half enter at a prolonged in man [12]. The 1.5 kg flavoprotein site with a potential above that of the NAD utilizes 20% of the total body oxygen consumption at couple. This results in the loss of the synthesis of about 1 rest, requiring 100–150 g of glucose per day [13]. of the 6 possible ATP molecules, for a loss of about 5% Although man can make about 10% of the glucose to in efficiency. The remaining reducing equivalents supply brain needs during fasting from fat [14], produced from the acetyl CoA produced during b prolonged in man leads to the excretion of oxidation, are metabolized in the TCA cycle in the 4–9 g of nitrogen per day which is equivalent to the normal fashion. However the redox potential of the Q destruction of 25–55 g /day. This amount of couple is not oxidized as it is during ketone metabolism, protein could supply between 17 and 32 g of but rather reduced, decreasing the DE available for ATP glucose per day, far below the 100–150 g/day required. synthesis. In addition, the elevation of free fatty acids, To supply the glucose required to support brain from leads to the increased transcription of mitochondrial protein alone would lead to death in about 10 days uncoupling proteins and of the enzymes of peroxisomal instead of the 57–73 days required to cause death in a b oxidation. Uncoupling proteins allow the proton young male of normal body composition during a total gradient generated by the respiratory chain to re-enter fast [15]. Cahill also noted that in his subjects under- the mitochondria by pathways which bypass the F1 going total starvation for 30 days, hunger subsided on ATPase generating heat rather than ATP. Fatty acids about the third day, coincident with the elevation of undergoing b oxidation with peroxisomes have no blood ketones to 7 mM. During prolonged fasting, the mechanism for energy conservation and result solely in human produces about 150 g of ketone bodies per day heat production. Induction of uncoupling proteins, by [16]. This suggests, that even though the Vmax of the chronic elevation of free fatty acids or from other causes monocarboxylate transporter in brain is increased result not in increased cardiac efficiency, but rather in during ketosis [17], the Km at the endothelial cell of pathological decreases in cardiac efficiency [10]. Among approximately 5 mM for ketone body transport of the common non-nitrogenous substrates for mitochon- approximately 5 mM would still dominate the ketone drial energy generation, ketone bodies deserve the effects in brain, so that a blood level only slightly below designation of a ‘‘superfuel’’. that of 7 mM ketone bodies would be required to ARTICLE IN PRESS 312 R.L. Veech / Prostaglandins, Leukotrienes and Essential Fatty Acids 70 (2004) 309–319 achieve the effects on brain metabolism were elevation review of the subject [21]. In a study of 600 patients of ketones to be achieved by means to achieve similar with a history of 20 seizures per day refractory to over 6 effects to those observed during prolonged starvation. drugs, the Hopkins group reported that the ketogenic These amounts are not pharmacological, but rather diet led to cessation of seizures in slightly less than one nutritional, which changes markedly the traditional third of subjects, a significant decrease in seizure pharmaceutical industry approaches to therapy. frequency in another third and no effect in one third Although brain has insulin receptors, it has either no [22]. Urinary ketone levels correlate poorly with blood or very low insulin levels making ketosis the only levels, so it is difficult to evaluate the effectiveness of the practical mechanism for increasing the efficiency of various ketogenic diets. One report of the blood levels of oxidation ATP generation in that organ. ketone bodies achieved on the diet suggests that blood Finally there are broad therapeutic implications from levels of about 4 mM are required for satisfactory results the ability of ketone body metabolism to oxidize the [23]. In a reverse of the increasing risk of a subsequent mitochondrial co-enzyme Q couple. The major source of seizure after the first one (the Gower effect), Freeman mitochondrial free radical generation is Q semiquinone reports that after 2 years on the ketogenic diet, a normal [18]. The semiquinone of Q, the half-reduced form, diet can be resumed without recurrence of seizures. spontaneously reacts with O2 to form free radicals. The mechanisms responsible for the therapeutic Oxidation of the Q couple reduces the amount of the response in remain unclear. Some authors have semiquinone form and thus would be expected to championed the effects of increased brain glutamate to dÀ decrease O2 production. In addition, the metabolism increase the inhibitory neurotransmitter GABA [24,25]. of ketones causes a reduction of the cytosolic free Others have attributed the therapeutic efficacy of the [NADP+]/[NADPH] couple which is in near-equili- ketogenic diet to a decrease in circulating blood glucose brium with the glutathione couple [19]. Reduced [26]. It is my hypothesis that the metabolism of ketones glutathione is the final reductant responsible for the in brain is likely to raise the DG0 of ATP hydrolysis, and + 2+ destruction of H2O2. with it the extent of the Na and Ca gradients which 0 From the multiple effects of the metabolism on the depend upon DGATP [27], thus raising the so-called basic pathways of intermediary metabolism, it is clear resting membrane potential and inhibiting the synchro- that there are a number of disease states in which mild nous neuronal discharge characteristic of epilepsy. ketosis could offer possible therapeutic benefits. Some of A number of variations of the Hopkins ketogenic diet these therapeutic uses of ketosis have been discussed have been reported, including those made up primarily earlier [20]. of mid chain length fatty acids [28] or diets containing large amounts of n-3 polyunsaturated fats [29]. In most variations, patient intolerance of the high fat diet is a 2. Ketogenic diets in human subjects major contributor to therapeutic failure. Also of concern is the elevation of blood which can Starvation, with attendant ketosis, has been used as a accompany a high fat diet. Freeman reports that the treatment for refractory epilepsy since the early 20th mean blood cholesterol on the Hopkins form of the diet, century. Pierre Marie proposed this treatment on the the average blood cholesterol rises to over 250, theory that epilepsy resulted from intestinal intoxica- significantly above generally recommended levels. In tion. On this assumption, a diet consisting of water only addition there are reports of dilated cardiomyopathy in for 30 days was used to successfully treat some patients on the ketogenic diet [30], which are not refractory epileptics by Hugh Conklin, a Wisconsin incompatible with the toxic effects of elevated plasma osteopath. The inference that ketone bodies themselves free fatty acids discussed earlier. Finally an increased were the effective agent, led Russell Wilder of the Mayo incidence in nephrolithiasis in patients on the ketogenic Clinic to propose a high fat, low diet for diet [31] as well as increases in serum uric acid secondary treatment of epilepsy. High fat, low carbohydrate diets to decreased urinary uric acid excretion have been were first used in medicine as a treatment for refractory reported in patients on a ketogenic diet. epilepsy. 2.2. Weight loss 2.1. Refractory epilepsy In the 1970s the ‘‘carbo-caloric diet’’ became popular, With the development, by Houston Merritt, of anti- advocating unlimited intake of all foodstuffs save epileptic drugs, use of the ketogenic diet fell into which were kept to specified limits. The disfavor. This approach to the treatment of drug high fat low carbohydrate diet produced both mild resistant epilepsy has continued to have its advocates, ketosis and weight loss, but unfortunately also produced most notably James Freeman and Elizabeth Vining of high levels of saturated plasma fats. As the public Johns Hopkins who have written a comprehensive became more conscious of the dangers of elevated ARTICLE IN PRESS R.L. Veech / Prostaglandins, Leukotrienes and Essential Fatty Acids 70 (2004) 309–319 313 and cholesterol, this diet program fell out combat casualties during the Viet Nam war when of favor. resuscitation with large volumes of conventional Ring- More recently, largely as the result of popularization er’s lactate became standard therapy. These studies in by Atkins [32], low fat high carbohydrate diets have animals suggest that fluids containing d,l-lactate are become popular again as an aid to weight loss. Studies associated with significant toxic effects which can be report significant weight loss on a high fat, high protein, avoided if d-b-hydroxybutyrate replaced the lactate. low carbohydrate diet without significant elevations of Progressive heart block ending in cardiac arrest can be serum cholesterol [33]. Studies comparing the so-called induced in rats by administration of Na d,l-lactate and Atkins diet versus more classical caloric restriction have similar forms of heart block are observed in children appeared recently and are the subject of significant receiving up to 20 l of Ringer’s d,l-lactate during the public interest. initial phases of burn therapy [41]. Investigation of the use of ketone containing fluids would seem indicated. 2.3. Adjuncts to cancer chemotherapy 3.2. Acyl CoA dehydrogenase deficiency As discussed earlier, elevation of ketone bodies decrease release from muscle as well as Perhaps the most dramatic therapeutic response to decreasing hepatic from amino acids. therapy with Na salts of ketone bodies has been the Feeding a ketogenic diet to mice with implanted tumors report of the response of 3 patients with the rare genetic decreased tumor size as well as decreasing muscle disease caused by acyl CoA dehydrogenase deficiency. wasting associated with tumor transplantation [34]. This disease is associated with an inability to metabo- Similar results have been reported in human cancer lized fatty acids by b oxidation lead to leukoencephalo- patients [35,36]. In human patients with advanced pathy and cardiomyopathy, of which the later form is astrocytomas, feeding of a ketogenic diet decreased more common. It is conventionally treated with a low tumor glucose uptake and in part of the group an fat diet and increased carnitine to bind free fatty acids. increase in patient performance [36]. In a surprising Three patients who became refractory to this therapy report in mice implanted with astrocytoma, a ketogenic were treated with very small doses in the order of 5 g/day diet associated with caloric restriction resulted in an of Na d,l-b-hydroxybutyrate and all showed dramatic 80% decrease in tumor mass and a decrease in tumor improvement in cardiac function and in the extent of vascularity implying an inhibition of angiogenesis [37]. leukodystrophy in the brain as judged by MRI [42]. One patient with quadriplegia showed a clearing of symp- toms, certainly a dramatic response. The l-form of b- 3. Salts of ketone bodies hydroxybutyrate is a product of b oxidation, while the d-form is the product of ketone body synthesis through The salts of ketone bodies have been examined for HMG CoA. for myelin production their therapeutic effects after either parenteral or oral requires the d-form for fatty acid synthesis, whereas administration. there would clearly be a deficiency of the l-form in this genetic condition. Which isomeric form is therapeutic in 3.1. Intravenous and dialysis fluid therapy this condition is unknown, since the administration of the d,l-form was dictated purely on the basis of cost of Traditional parenteral fluid therapy as currently the material from Sigma, which is dependent not on practiced uses fluids whose compositions are historical inherent cost, but rather on commercial availability. and have not been subjected to systematic study of less An important observation in this and earlier studies toxic alternatives. It has been argued that the use of was that the administration of Na d,l-b-hydroxybuty- acetate in dialysis fluids and racemic d,l-lactate in both rate lowered markedly the blood levels of free fatty acids dialysis and parenteral fluid therapy for burns or from 0.6 mM to less than 0.1 mM. Oral administration resuscitation results in significant and unnecessary of Na d,l-b-hydroxybutyrate raised blood levels of total toxicity [38]. A recent report by the Academy of ketones to 0.4 mM at 1 h, a very low level relative to the Medicine suggests a more systematic investigation of Km for brain endothelial transport of 5 mM, but similar the use of Na d-b-hydroxybutyrate as a replacement of to the Km for neuronal or mitochondrial transport of the conventional anions in parenteral fluid therapy [39]. 0.5 mM. The Km for transport remain un- In response, studies funded by the military have known, but is clearly a pressing issue if one is to suggested that resuscitation fluids containing Na d-b- understand how such low levels of ketone bodies can hydroxybutyrate instead of the traditional d,l-lactate lower the blood levels of free fatty acids by over 75%. can prevent apoptosis in lung which occurs 24 h after The lowering of release of free fatty acid from adi- severe hemorrhage in a rodent model [40]. DaNang lung pocytes into blood, cannot be simply ascribed to simple was a leading cause of morbidity and mortality in ‘‘end product’’ inhibition since it is not reasonable to ARTICLE IN PRESS 314 R.L. Veech / Prostaglandins, Leukotrienes and Essential Fatty Acids 70 (2004) 309–319 believe that an adipocyte process of free fatty acid visceral obesity and hypertension [44]. Insulin resistance release is simply enzymatically related to the hepatic is present in obese subjects and occurs during ‘‘stressful’’ process of ketone body production. Other factors such conditions characterized by elevated adrenal as the effects of ketosis upon kinase activity or and catechol amines. Insulin resistance also occurs in the activity of the adipocyte hormone sensitive lipase conditions in which inflammatory cytokines are ele- must be at play here. Elucidation of the effects of vated. Acute insulin resistance is seen in alcohol abusers ketones upon free fatty acid release from is where elevation of either 2,3 butandiol or 1,2 propandiol an important and unexplored area. inhibits insulin action on adipocytes [45] and impair whole body glucose utilization [46]. Given the metabolic effects of insulin, it is reasonable to suppose that mild 4. Animal and cellular studies of ketone bodies in disease ketosis might offer a therapeutic potential which acts models directly on the primitive metabolic pathways themselves without requiring the action of the complex insulin 4.1. Insulin resistant states signaling pathway. The most extreme example of insulin resistance is The excessive production of ketone bodies during Leprechaunism and Rabson-Mendenhall syndrome, diabetic is a life threatening condition rare genetic diseases where a mutation in the insulin usually seen in type I diabetics after some intercurrent receptor gene results in the loss of insulin binding [47]. event. It is characterized by profound hyperglycemia Some therapeutic success has resulted from treatment with insulin resistance and elevated blood ketone bodies with insulin like growth factor [48], but this material is À approaching 25 mM, blood HCO3 approaching 0 and not widely available. Currently these children are treated blood pH approaching 7 causing hyperventilation and a with huge doses of insulin which, without effective compensatory low pCO2. Death occurs from the low pH insulin receptors, are without effect, and merely reflect and vascular collapse secondary to urinary loss of Na+ the physician’s impotence in the face of a devastating and K+ in an osmotic . It is not surprising that condition. Death usually occurs in these children in, or physicians view elevation of blood ketone bodies with before late adolescence. In the absence of effective alarm. available treatment, mild ketosis might offer therapeutic However, our data on the acute effects of insulin and benefits to these children who are currently without ketone bodies in the perfused working heart suggest that effective therapy. ketosis, within limits, mimic the acute effects of insulin [43]. Insulin has two major acute effects in heart. It increases intracellular glucose concentrations by causing 4.2. Genetic defects in glucose transport and PDH the movement of the glucose transporter, GLUT4, from activity endoplasmic reticulum to the plasma membrane while at the same time increasing the mitochondrial acetyl CoA While insulin resistance is associated with hormonally concentrations by increasing the activity of the pyruvate induced defects in both glucose transport and PDH dehydrogenase multienzyme complex. Ketone bodies activity, specific genetic defects in both of these increase the intracellular glucose concentration by activities, while rare, have been extensively studied. providing an alternative metabolic substrate and they Glucose transport across endothelial cells forming the increase mitochondrial acetyl CoA concentration by by- blood brain barrier is accomplished by GLUT1. passing the PDH complex and instead providing acetyl GLUT1 is also present in glia other than microglia CoA from acetoacetyl CoA. Both insulin and ketones which express GLUT5, while transport into neurons is a have the same effects on the metabolites of the first third function of GLUT3 [49]. Autosomal dominant genetic to the citric acid cycle, on mitochondrial redox states defects in GLUT1, usually associated with infantile and both increase the hydraulic efficiency of the working epilepsy, result in a generalized energy deficit in brain perfused heart. Viewed in this light, mild ketosis and are successfully treated with the ketogenic diet [50]. provides the same metabolic effects as insulin, but at Leigh’s syndrome is the most frequent metabolic the metabolic or primitive control level which by-passes disorder in infancy characterized by lactic and the complex signaling pathway of insulin. During bilateral encephalopathy usually involving the white prolonged fasting, when insulin levels approach 0, mild matter of the substantia nigra and medulla oblongata. ketosis compensates metabolically for the absence of Classically, this condition was associated with defects in insulin effects. It follows that the induction of mild multienzyme complex. As such, ketosis would be therapeutic in insulin resistant states. it should be treatable with mild ketosis. Unfortunately Insulin resistant states are extremely common. Insulin the clinical phenotype of Leigh’s syndrome can occur resistance is the hallmark of type II and the so- with multiple defects in the electron transport system called ‘‘’’ where it is associated with and in the F1 ATPase. The therapeutic response of these ARTICLE IN PRESS R.L. Veech / Prostaglandins, Leukotrienes and Essential Fatty Acids 70 (2004) 309–319 315 heterogeneous types of defects would naturally be following a stroke. In areas of tissue, where O2 supply is mixed. limited, the provision of ketone bodies might provide benefit in limiting the area of damage. In a neonatal 4.3. Hypoglycemic episodes rodent model of hypoxia, induction of ketosis has been reported to limit brain damage in comparison to control A major limitation in achieving ‘‘tight control’’ of animals after exposure to 3 h of hypoxia [53]. Similar diabetics, is the risk of increased episodes of hypogly- effects of ketone bodies in limiting the area of brain cemia. This consequence of robust insulin therapy is damage have been reported in rodent bilateral carotid particularly frequent in type I diabetics. Because of the occlusion models [54]. potentially serious consequences of cognitive impair- No studies have been undertaken to determine if ment associated with , physicians are ketosis might be effective in the treatment of angina reluctant to keep blood glucose within ranges which pectoris or in increasing the walking distance in the are thought to be optimum for the prevention of long- claudication associated with peripheral vascular disease. term vascular disease. Ketone bodies are an alternative From a theoretical point of view, one might expect to glucose as a supplier of the metabolic energy needs benefits from ketosis in these common conditions. for brain. Cahill has shown [51] that during prolonged Unfortunately, aside from parenteral infusion or oral fasting, when total blood ketone bodies are in the 5– ingestion of ketone and their salts, it is not possible at 7 mM range, blood glucose concentrations can be present to elevation ketone bodies, which might improve decreased to below 1 mM without either convulsions metabolic efficiency without increasing free fatty acids or any discernable impairment of cognitive function. At which would be expected to decrease metabolic effi- these concentrations, ketone bodies can provide essen- ciency for reasons discussed previously. tially all of the energy demands in brain to maintain function. The induction of mild ketosis therefore offers a 4.5. Muscle wasting method for obtaining tighter control of blood glucose in brittle diabetics without the induction of the physiolo- During starvation, ketosis prevents the destruction of gical consequences of hypoglycemia on cerebral func- muscle mass and decreases the export of alanine and tion. glutamine from muscle by decreasing the gluconeogenic demands to provide glucose to brain. One might 4.4. Hypoxic states therefore expect that ketosis would prevent muscle wasting following surgery or trauma. Unfortunately The metabolism of ketone bodies results in a 28% existing evidence of the effects of the infusion of Na d-b- increase in the hydraulic work produced by perfused hydroxybutyrate in patients with septic shock found no heart per unit of O2 consumed when compared with the effect on the degree of muscle wasting [55]. The reason metabolism of glucose alone. There are many conditions for this apparently inexplicable observation may be where limitation of O2 supply results in pathology. found in observations by Murad and his group [56] Ketone bodies would, of course, be expected to have no where they showed that administration of endotoxin effect in conditions of total anoxia, since they require results in nitration and inactivation of 3-oxoacid CoA mitochondrial electron transport to exert their effects. transferase, the enzyme required to activate ketones However in conditions of hypoxia, or relative O2 lack making their metabolic utilization possible. An increase the induction of ketosis might be expected to offer some in NO production and hence nitration of this benefit. enzyme may account for a number of conditions where One non-medical use in this category would be the use ketone body utilization may be decreased. of mild ketosis to improve physical performance in settings where extreme exertion is required. Such 4.6. Genetic myopathies situations would exist in the military when troops are under extreme combat stress and in certain civilian Friedreich’s ataxia is the most common hereditary settings involving emergency personnel. Extreme exer- limb and gait ataxia associated with hypertrophic tion leads not only to exhausting but also to impairment cardiomyopathy. It is an autosomal recessive disease of cognitive and motor skills under conditions of caloric resulting from GAA repeats in the gene’s first intron restriction [52]. Mild ketosis may offer a way to increase which impairs the transcription of the nuclear encoded muscle and brain function without elevation of free fatty mitochondrial protein frataxin leading to dysregulation acids which will decrease muscle and cardiac metabolic of iron homeostasis with impairment of the activity of efficiency through the induction of mitochondrial mitochondrial iron–sulfur containing proteins such as uncoupling protein. aconitase and some of the respiratory chain complexes. Obvious in this category would be the penumbral While yeast frataxin binds iron, the human form of this damage in heart after coronary occlusion or in brain protein does not [57]. The precise mechanism whereby ARTICLE IN PRESS 316 R.L. Veech / Prostaglandins, Leukotrienes and Essential Fatty Acids 70 (2004) 309–319 decrease in frataxin impairs mitochondrial function is cytosolic [NADP+]/[NADPH] couple [43] whose near not precisely known. It is known however that cardiac equilibrium substrate glutathione is the terminal dis- phosphocreatine is decreased and inorganic phosphorus tructant of H2O2. An instant form of Parkinsonism can increased in the of patients with Friedreich’s be induced in humans, animals or cells by administra- ataxia indicating a deficiency in cardiac ATP production tion of the meperidine analogue and free radical [58]. It also is known that aconitase activity is impaired generator MPP+ [60,61]. Administration of 4 mM d-b- and that over expression of frataxin in mammalian cells hydroxybutyrate to mesencephalic dopaminergic pri- results in increased TCA cycle flux, increased mitochon- mary neuronal cultures rescues these neurons from drial membrane potential and increased ATP content death induced by MPP+ [62]. These findings suggest [59]. In our studies of the effects of ketone bodies in that mild ketosis might be an effective therapy in this heart, ketone body metabolism increased the heart disease. citrate content 3 fold which would be expected to decrease a block in aconitase activity which converts 4.8. Neurodegenerative diseases citrate to isocitrate. Ketones also increase the DG0 of ATP and thus should ameliorate the defects observed in Alzheimer’s disease is a common form of dementia the hearts of these patients. Antioxidant therapies of affecting 3% of those 65–74 years old, 18.7% of those various sorts have been reported to improve cardiac 75–84, and 47.2% of those over 85 years of age [63]. function in these patients, but so far have had no effect Approximately 20–30% of Alzheimer’s disease results in on the neurological impairment. One might expect mild defects in 6 different genes. Defects in chromosome 1 ketosis to improve the function in both heart and brain. and 14, encoding presenilin 1 and 2; in chromosome 21, encoding amyloid precursor protein, lead to early onset 4.7. Diseases of free radical toxicity Alzheimer’s disease. Defects in chromosome 21, encod- ing a2 microglobulin; or in chromosome 19, encoding Parkinson’s disease is a common, generally acquired apolipoprotein E is associated with late onset Alzhei- movement disorder characterized by bradykinesia, mer’s disease. All of the genetic defects result in rigidity and tremor resulting from destruction of over excessive accumulation of amyloid protein. The remain- 80% of the mesencephalic dopaminergic neurons when ing 70–80% of Alzheimer’s disease have the same clinical symptoms of the disease appear. Dopaminergic pathological findings of amyloid plaques, phosphory- neurons decline in numbers in all aging humans, but the lated microtubular proteins and loss of hippocampal rate of destruction is accelerated in patients with clinical neurons associated with memory loss and increasing Parkinsonism, with symptoms most commonly appear- dementia, but from causes which include ischemia [64] ing in the 6th decade. It is thought to result from mild trauma [65] elevated plasma homocystine [66] continued free radical damage to dopaminergic neurons, insulin resistance [67] and impaired brain energy which with high iron content, are particularly subject to metabolism [68]. The clinical phenotype of Alzheimer’s this toxicity. The disease is treatable for a time by dopa disease is clearly a complex multifactoral disease. administration, but as damage and death of dopami- A major finding in Alzheimer’s disease is the decrease nergic neurons continues, dopa therapy becomes either in brain acetyl choline [69]. This finding has led to the ineffective or limited by its toxicity. Since there is little widespread use of acetyl cholinesterase inhibitor in the genetic loading in this disease, except in the rare early treatment of the disease, but with only very limited if onset forms, it is clear that non-genetic therapies are any improvement [70]. However this pharmacological essential. approach fails to address the underlying pathophysiol- The major sources of free radical production is the ogy of the disease. The common feature of Alzheimer’s partially reduced co-enzyme Q semiquinone [18]. The disease is the elevated levels of proteolytic fragments of semiquinone spontaneously reacts with O2 to form b amyloid both extra and intracellularly [71].Itis dÀ superoxide anion in the reaction: QH +O2- reported that the 1–42 fragment of b amyloid, Ab1À42, dÀ + O2 +H . A reaction catalyzed by superoxide dismu- stimulates a mitochondrial isoform of glycogen synthase dÀ dÀ + tase, can form H2O2:O2 +O2 +2H -O2+H2O2. kinase 3b [72] which phosphorylates and inactivates the Hydrogen peroxide can either be destroyed by glu- pyruvate dehydrogenase multienzyme complex [73] and tathione: 2GSH+H2O2-GSSG+2H2O, or in the results in the decrease in acetyl choline synthesis [74] presence of iron, as occurs in dopaminergic neurons characteristic of the Alzheimer’s disease phenotype. can form the more toxic hydroxyl radical in the so-called Since under normal conditions brain is entirely depen- 2+ 3+ Fenton reaction: H2O2+Fe -HO+OH+Fe . The dent on glucose as an energy source, inhibition of PDH metabolism of ketone bodies oxidizes the Q couple and would be expected to decrease mitochondrial acetyl therefore should decrease the amount of Q semiquinone, CoA formation and hence citrate formation, a necessary QHdÀ, thereby decreasing free radical production. In precursor of acetyl choline. Blockade of PDH is addition the metabolism of ketones, reduces the characteristic of insulin lack in heart and ketone bodies ARTICLE IN PRESS R.L. Veech / Prostaglandins, Leukotrienes and Essential Fatty Acids 70 (2004) 309–319 317 are the physiological mechanism which overcomes this therapeutic blood levels without either the gastric inhibition. If this were to occur in neurons, administra- intolerance or hyperlipidemic effects of high fat diets. tion of ketone bodies should by-pass this block. We tested this hypothesis in primary rat hippocampal neuronal cultures exposed to 5 mMAb1À42 and found References that the addition of 4 mM Na d-b-hydroxybutyrate [1] Y. Kashiwaya, K. Sato, N. Tsuchiya, S. Thomas, D.A. Fell, R.L. protected against Ab1À42 toxicity [62]. Induction of mild ketosis would therefore seem a reasonable potential Veech, et al., Control of glucose utilization in working perfused rat heart, J. Biol. Chem. 269 (1994) 25502–25514. therapy in Alzheimer’s disease. [2] K. Sato, Y. Kashiwaya, C.A. Keon, N. Tsuchiya, M.T. King, G.K. Radda, et al., Insulin, ketone bodies, and mitochondrial energy transduction, FASEB J. 9 (1995) 651–658. 4.9. Methods of induction of ketosis [3] D.F. Horrobin, Innovation in the pharmaceutical industry, J. R. Soc. Med. 93 (7) (2000) 341–345. [4] H.A. Krebs, R.L. Veech, Pyridine nucleotide interrelations, in: S. Mild ketosis can be induced in man by either Papa, J.M. Tager, E. Quagliariello, E.C. Slater (Eds.), The Energy prolonged fasting or by feeding a high fat, low Level and Metabolic Control in Mitochondria, Bari, Adriatica carbohydrate diet. Total starvation is not a therapeutic Editrice, 1969, pp. 329–382. option, since death results in a normal weight man in [5] W.M. Clark, Oxidation Reduction Potentials of Organic Systems, 68–72 days. Feeding a high fat, low carbohydrate diet in Williams and Wilkins Co., Baltimore, 1960. [6] P. Mitchell, Chemiosmotic Coupling and Energy Transduction, adults can result in elevation of or choles- Glynn Research Ltd., Bodmin, 1968. terol or both. The vascular pathology accompanying [7] C. Gibbons, M.G. Montgomery, A.G.W. Leslie, J.E. Walker, The these changes limits the use of such diets, particularly in structure of the central stalk in bovine F1-ATPase at 2.4 A the elderly. Attempts to design high fat diet enriched resolution, Nature Struct. Biol. 7 (2000) 1055–1061. with polyunsaturated or short chain fatty acids may [8] K. Yasuda, H. Noji, K. Kinosita Jr., M. Yoshida, F1-ATPase is a highly efficient molecular motor that rotates with discrete 120 avoid elevation of cholesterol levels, but suffer from steps, Cell 93 (1998) 1117–1124. poor patient tolerance of the diet. [9] D.H. Williamson, P. Lund, H.A. Krebs, The redox state of free Oral administration of Na d,l-b-hydroxybutyrate in -adenine dinucleotide in the cytoplasm and mito- doses from 80 to 900 mg/kg/day was sufficient to achieve chondria of rat liver, Biochem. J. 103 (1967) 514–527. peak blood levels of total d-b-hydroxybutyrate+ace- [10] E.A. Boehm, B.E. Jones, G.K. Radda, R.L. Veech, K. Clarke, Increased uncoupling proteins and decreased efficiency in toacetate of 0.19–0.36 mM producing a therapeutic palmitate-perfused hyperthyroid rat heart, Am. J. Physiol Heart response in children with acyl CoA dehydrogenase Circ. Physiol 280 (3) (2001) H977–H983. deficiency [42]. In a 70 kg man to achieve these levels [11] O.E. Owen, A.P. Morgan, H.G. Kemp, J.M. Sullivan, M.G. of ketosis would require the feeding of between 5.6 to Herrera, G.F. Cahill Jr., Brain metabolism during fasting, J. Clin. 63 g/day at a present cost of $3 gÀ1 or $17 to $189 dayÀ1. Invest. 46 (1967) 1589–1595. [12] G.F. Cahill Jr., Starvation in man, N. Engl. J. Med. 282 (1970) In normal fasting man achieving blood levels of 5–7 mM 668–675. total ketone bodies, the daily production of ketone [13] G.F. Cahill Jr., M.G. Herrera, A.P. Morgan, J.S. Soeldner, bodies is about 150 g per day [16]. It is believed that to J. Steinke, P.L. Levy, et al., Hormone-fuel interrelationships achieve a therapeutic response in refractory epilepsy, the during fasting, J. Clin. Invest 45 (11) (1966) 1751–1769. level of blood ketone should be above 4 mM [23] which [14] J.P. Casazza, M.E. Felver, R.L. Veech, The metabolism of in rat, J. Biol. Chem. 259 (1984) 231–236. is compatible with a Km for ketone body transport by [15] T.B. VanItallie, T.H. Nufert, Ketones: metabolism’s ‘‘Ugly the monocarboxylate transporter into brain of about Duckling’’, Annu. Rev. Nutr. 61 (10) (2003) 327–341. + 5 mM. Leaving aside the effects of such a large Na [16] G.A. Reichard, O.E. Owen, A.C. Haff, P. Paul, W.M. Bortz, load, the present costs of the administration of salts of Ketone-body production and oxidation in fasting obese humans, d-b-hydroxybutyrate to achieve ketosis makes this J. Clin. Invest. 53 (2) (1974) 508–515. [17] R.L. Leino, D.Z. Gerhart, R. Duelli, B.E. Enerson, L.R. Drewes, approach unlikely. Diet-induced ketosis increases monocarboxylate transporter Poly d-b-hydroxybutyrate occurs at up to 90% dry (MCT1) levels in rat brain, Neurochem. Int. 38 (6) (2001) weight in certain microbial species [75] and has been 519–527. produced in commercial quantities as a biodegradable [18] B. Chance, H. Sies, A. Boveris, Hydroperoxide metabolism in substitute for polyethylene at a cost of less than $2 per mammalian organs, Physiol. Rev. 59 (3) (1979) 527–605. d [19] R.L. Veech, L.V. Eggleston, H.A. Krebs, The redox state of free kg. Poly -b-hydroxybutyrate also occurs in mamma- nicotinamide-adenine dinucleotide phosphate in the cytoplasm of lian species in both blood plasma and in mitochondrial rat liver, Biochem. J. 115 (1969) 609–619. membranes [76]. The bacterial poly d-b-hydroxybuty- [20] R.L. Veech, B. Chance, Y. Kashiwaya, H.A. Lardy, G.F. Cahill rate contains 200–500,000 monomeric units and is not Jr., Ketone bodies, potential therapeutic uses, IUBMB Life 51 (4) readily degradable in mammalian gut. However, pre- (2001) 241–247. [21] J.M. Freeman, E.P.G. Vining, Intractable epilepsy, Epilepsia 33 paration of hydrolysis products of this material into (1992) 1132–1136. metabolizable forms offers promise that oral or par- [22] S.L. Kinsman, E.P. Vining, S.A. Quaskey, D. Mellits, J.M. enteral administration of ketone esters may achieve Freeman, Efficacy of the ketogenic diet for intractable ARTICLE IN PRESS 318 R.L. Veech / Prostaglandins, Leukotrienes and Essential Fatty Acids 70 (2004) 309–319

seizure disorders: review of 58 cases, Epilepsia 33 (6) (1992) [43] Y. Kashiwaya, M.T. King, R.L. Veech, Substrate signaling by 1132–1136. insulin: a ketone bodies ratio mimics insulin action in heart, Am. [23] D.L. Gilbert, P.L. Pyzik, J.M. Freeman, The ketogenic diet: J. Cardiol. 80 (3A) (1997) 50A–64A. seizure control correlates better with serum beta-hydroxybutyrate [44] P. Bjorntorp, G. Holm, R. Rosmond, B. Folkow, Hypertension than with urine ketones, J. Child Neurol. 15 (12) (2000) 787–790. and the metabolic syndrome: closely related central origin? Blood [24] M. Erecinska, D. Nelson, Y. Daikhin, M. Yudkoff, Regulation of Pressure 9 (2–3) (2000) 71–82. GABA level in rat brain synaptosomes: fluxes through enzymes of [45] F. Lomeo, M.A. Khokher, P. Dandona, Ethanol and its novel the GABA shunt and effects of glutamate, calcium, and ketone metabolites inhibit insulin action on adipocytes, Diabetes 37 (7) bodies, J. Neurochem. 67 (6) (1996) 2325–2334. (1988) 912–915. [25] M. Yudkoff, Y. Daikhin, I. Nissim, R. Grunstein, Effects of [46] D. Xu, A.S. Dhillon, A. Abelmann, K. Croft, T.J. Peters, T.N. ketone bodies on astrocyte amino acid metabolism, J. Neuro- Palmer, Alcohol-related diols cause acute insulin resistance chem. 69 (2) (1997) 682–692. in vivo, Metabolism 47 (10) (1998) 1180–1186. [26] A.E. Greene, M.T. Todorova, R. McGowan, T.N. Seyfried, [47] P. Roach, Y. Zick, P. Formisano, D. Accili, S.I. Taylor, P. Caloric restriction inhibits seizure susceptibility in epileptic Gorden, A novel human insulin receptor gene mutation uniquely EL mice by reducing blood glucose, Epilepsia 42 (11) (2001) inhibits insulin binding without impairing posttranslational 1371–1378. processing, Diabetes 43 (9) (1994) 1096–1102. [27] R.L. Veech, Y. Kashiwaya, D.N. Gates, M.T. King, K. Clarke, [48] C.A. Bondy, L.E. Underwood, D.R. Clemmons, H.P. Guler, The energetics of ion distribution: the origin of the resting electric M.A. Bach, M. Skarulis, Clinical uses of insulin-like growth potential of cells, IUBMB Life 54 (2002) 241–252. factor I [see comments], Ann. Intern. Med. 120 (7) (1994) [28] P.R. Huttenlocher, A.J. Wilbourn, J.M. Signore, Medium-chain 593–601. triglycerides as a therapy for intractable childhood epilepsy, [49] S.J. Vannucci, F. Maher, I.A. Simpson, Glucose transporter Neurology 21 (11) (1971) 1097–1103. proteins in brain: delivery of glucose to neurons and glia, Glia 21 [29] C.A. Dell, S.S. Likhodii, K. Musa, M.A. Ryan, W.M. Burnham, (1) (1997) 2–21. S.C. Cunnane, Lipid and fatty acid profiles in rats consuming [50] D.C. De Vivo, L. Leary, D. Wang, Glucose transporter 1 different high-fat ketogenic diets, 36 (4) (2001) 373–378. deficiency syndrome and other glycolytic defects, J. Child Neurol. [30] T.H. Best, D.N. Franz, D.L. Gilbert, D.P. Nelson, M.R. Epstein, 17 (Suppl 3) (2002) 3S15–23 (discussion 3S24–5:3S15–23). Cardiac complications in pediatric patients on the ketogenic diet, [51] G.F. Cahill Jr., T.T. Aoki, Alternate fuel utilization in brain, in: Neurology 54 (12) (2000) 2328–2330. J.V. Passonneau, R.A. Hawkins, W.D. Lust, F.A. Welsh (Eds.), [31] S. Kielb, H.P. Koo, D.A. Bloom, G.J. Faerber, Nephrolithiasis Cerebral Metabolism and Neural Function, Williams & Wilkins, associated with the ketogenic diet, J. Urol. 164 (2) (2000) 464–466. Baltimore, 1980, pp. 234–242. [32] G. Taubes, What if its all been a Big Fat Lie? Sect. Magazine, NY [52] P.N. Ainslie, I.T. Campbell, K.N. Frayn, S.M. Humphreys, D.P. Times, 2002. MacLaren, T. Reilly, Physiological, metabolic, and performance [33] E.C. Westman, W.S. Yancy, J.S. Edman, K.F. Tomlin, C.E. implications of a prolonged hill walk: influence of energy intake, Perkins, Effect of 6-month adherence to a very low carbohydrate J. Appl. Physiol. 94 (3) (2003) 1075–1083. diet program, Am. J. Med. 113 (1) (2002) 30–36. [53] B.J. Dardzinski, S.L. Smith, J. Towfighi, G.D. Williams, R.C. [34] S.A. Beck, M.J. Tisdale, Nitrogen excretion in cancer cachexia Vannucci, M.B. Smith, Increased plasma beta-hydroxybutyrate, and its modification by a high fat diet in mice, Cancer Res. 49 (14) preserved cerebral energy metabolism, and amelioration of brain (1989) 3800–3804. damage during neonatal hypoxia ischemia with dexamethasone [35] L.C. Nebeling, E. Lerner, Implementing a ketogenic diet based on pretreatment, Pediatr. Res. 48 (2) (2000) 248–255. medium-chain triglyceride oil in pediatric patients with cancer, J. [54] M. Suzuki, M. Suzuki, K. Sato, S. Dohi, T. Sato, A. Matsuura, Am. Diet. Assoc. 95 (6) (1995) 693–697. et al., Effect of beta-hydroxybutyrate, a cerebral function [36] L.C. Nebeling, F. Miraldi, S.B. Shurin, E. Lerner, Effects of a improving agent, on cerebral hypoxia, anoxia and ischemia in ketogenic diet on tumor metabolism and nutritional status in mice and rats, Jpn. J. Pharmacol. 87 (2) (2001) 143–150. pediatric oncology patients: two case reports, J. Am. Coll. Nutr. [55] M. Beylot, D. Chassard, C. Chambrier, M. Guiraud, M. Odeon, 14 (2) (1995) 202–208. B. Beaufrere, et al., Metabolic effects of a d-beta-hydroxybutyrate [37] P. Mukherjee, M.M. El Abbadi, J.L. Kasperzyk, M.K. Ranes, infusion in septic patients: inhibition of and glucose T.N. Seyfried, Dietary restriction reduces angiogenesis and production but not leucine oxidation, Crit. Care Med. 22 (7) growth in an orthotopic mouse brain tumour model, Br. J. (1994) 1091–1098. Cancer 86 (10) (2002) 1615–1621. [56] S. Marcondes, I.V. Turko, F. Murad, Nitration of succinyl- [38] R.L. Veech, The toxic impact of parenteral solutions on the CoA:3-oxoacid CoA-transferase in rats after endotoxin adminis- metabolism of cells: a hypothesis for physiological parenteral tration, Proc. Natl. Acad. Sci. USA 98 (13) (2001) 7146–7151. therapy, Am. J. Clin. Nutr. 44 (1986) 519–551. [57] S. Adinolfi, M. Trifuoggi, A.S. Politou, S. Martin, A. Pastore, A [39] Fluid Resuscitation: State of the Science for Treating Combat structural approach to understanding the iron-binding properties Casualties and Civilian Injuries, National Academy Press, of phylogenetically different frataxins, Hum. Mol. Genet. 11 (16) Washington DC, 1999. (2002) 1865–1877. [40] H.B. Alam, B. Austin, E. Koustova, P. Rhee, Resuscitation- [58] M. Bunse, N. Bit-Avragim, A. Riefflin, A. Perrot, O. Schmidt, induced pulmonary apoptosis and intracellular adhesion mole- F.R. Kreuz, et al., Cardiac energetics correlates to myocardial cule-1 expression in rats are attenuated by the use of Ketone hypertrophy in Friedreich’s ataxia, Ann. Neurol. 53 (1) (2003) Ringer’s solution, J. Am. Coll. Surg. 193 (3) (2001) 255–263. 121–123. [41] L. Chan, J. Slater, J. Hasbargen, D.N. Herndon, R.L. Veech, S. [59] M. Ristow, M.F. Pfister, A.J. Yee, M. Schubert, L. Michael, C.Y. Wolf, Neurocardiac toxicity of racemic d,l-lactate fluids, Integr. Zhang, et al., Frataxin activates mitochondrial energy conversion Physiol. Behav. Sci. 29 (1994) 383–394. and oxidative phosphorylation [In Process Citation], Proc. Natl. [42] J.L. Van Hove, S. Grunewald, J. Jaeken, P. Demaerel, P.E. Acad. Sci. USA 97 (22) (2000) 12239–12243. Declercq, P. Bourdoux, et al., d,l-3-hydroxybutyrate treatment [60] J.W. Langston, E.B. Langston, I. Irwin, MPTP-induced parkin- of multiple acyl-CoA dehydrogenase deficiency (MADD), Lancet sonism in human and non-human primates—clinical and experi- 361 (9367) (2003) 1433–1435. mental aspects, Acta Neurol. Scand. Suppl. 100 (1984) 49–54. ARTICLE IN PRESS R.L. Veech / Prostaglandins, Leukotrienes and Essential Fatty Acids 70 (2004) 309–319 319

[61] J.D. Adams Jr., L.K. Klaidman, A.C. Leung, MPP+ and [69] A. Pope, H.H. Hess, E. Lewin, Microchemical pathology of the MPDP+ induced oxygen radical formation with mitochondrial cerebral cortex in presenile dementia, Trans. Am. Neurol. Assoc. enzymes, Free Radic. Biol. Med. 5 (2) (1993) 181–186. 89 (1965) 15–16. [62] Y. Kashiwaya, T. Takeshima, N. Mori, K. Nakashima, K. [70] T.W. Robbins, G. McAlonan, J.L. Muir, B.J. Everitt, Cognitive Clarke, R.L. Veech, d-beta-hydroxybutyrate protects neurons in enhancers in theory and practice: studies of the cholinergic models of Alzheimer’s and Parkinson’s disease, Proc. Natl. Acad. hypothesis of cognitive deficits in Alzheimer’s disease, Behav. Sci. USA 97 (10) (2000) 5440–5444. Brain Res. 83 (1–2) (1997) 15–23. [63] D.A. Evans, H.H. Funkenstein, M.S. Albert, P.A. Scherr, N.R. [71] Y.M. Kuo, M.R. Emmerling, C. Vigo-Pelfrey, T.C. Kasunic, J.B. Cook, M.J. Chown, et al., Prevalence of Alzheimer’s disease in a Kirkpatrick, G.H. Murdoch, et al., Water-soluble Abeta (N-40, community population of older persons. Higher than previously N-42] oligomers in normal and Alzheimer disease , J. Biol. reported [see comments], J. Am. Med. Assoc. 262 (18) (1989) Chem. 271 (8) (1996) 4077–4081. 2551–2556. [72] M. Hoshi, M. Sato, S. Kondo, A. Takashima, K. Noguchi, M. [64] R.N. Kalaria, S.U. Bhatti, E.A. Palatinsky, D.H. Pennington, Takahashi, et al., Different localization of tau protein kinase I/ E.R. Shelton, H.W. Chan, et al., Accumulation of the beta glycogen synthase kinase- 3 beta from glycogen synthase kinase-3 amyloid precursor protein at sites of ischemic injury in rat brain, alpha in cerebellum mitochondria, J. Biochem. 118 (4) (1995) Neuroreport 4 (2) (1993) 211–214. 683–685 (Tokyo). [65] G. Kanayama, M. Takeda, H. Niigawa, Y. Ikura, H. Tamii, N. [73] M. Hoshi, A. Takashima, K. Noguchi, M. Murayama, M. Sato, S. Taniguchi, et al., The effects of repetitive mild brain injury on Kondo, et al., Regulation of mitochondrial pyruvate dehydrogen- cytoskeletal protein and behavior, Methods Find. Exp. Clin. ase activity by tau protein kinase I/glycogen synthase kinase 3beta Pharmacol. 18 (2) (1996) 105–115. in brain, Proc. Natl. Acad. Sci. USA 93 (7) (1996) 2719–2723. [66] S. Seshadri, A. Beiser, J. Selhub, P.F. Jacques, I.H. Rosenberg, [74] M. Hoshi, A. Takashima, M. Murayama, K. Yasutake, N.

R.B. D’Agostino, et al., Plasma homocysteine as a risk factor for Yoshida, K. Ishiguro, et al., Nontoxic amyloid b peptide1À42 dementia and Alzheimer’s disease, N. Engl. J. Med. 346 (7) (2002) suppresses acetylcholine synthesis, J. Biol. Chem. 272 (4) (1997) 476–483. 2038–2041. [67] J. Kuusisto, K. Koivisto, L. Mykkanen, F.L. Helkala, M. [75] H.M. Muller, D. Seebach, Poly(hydroxyalkanoates): a fifth class Vanhunen, K. Kervinen, et al., Association between features of of physiologically important organic biopolyers, Angew. Chem. the insulin resistance syndrome and Alzheimer’s disease indepen- 32 (1994) 477–502. dently of apolipoprotein E4 phenotype: cross sectional population [76] D. Seebach, A. Brunner, H.M. Burger, J. Schneider, R.N. Reusch, based study, Brit. Med. J. 315 (1997) 1045–1049. Isolation and 1H-NMRspectroscopic identification of poly(3- [68] D. Gabuzda, J. Busciglio, L.B. Chen, P. Matsudaira, B.A. hydroxybutanoate) from prokaryotic and eukaryotic organisms. Yankner, Inhibition of energy metabolism alters the processing Determination of the absolute configuration (R) of the mono- of amyloid precursor protein and induces a potentially amyloido- meric unit 3-hydroxybutanoic acid from Escherichia coli and genic derivative, J. Biol. Chem. 269 (1994) 13623–13628. spinach, Eur. J. Biochem. 224 (2) (1994) 317–328.